Method and apparatus for encapsulation of biologically-active substances in cells

ABSTRACT

The present invention relates to a method and apparatus for the encapsulation of biologically-active substances in a red blood cell, characterized by an optionally automated, continuous-flow, self-contained electroporation system which allows withdrawal of blood from a patient, separation of red blood cells, encapsulation of a biologically-active substance in the cells, and optional recombination of blood plasma and the modified cells, thereby producing blood with modified biological characteristics. The present invention is particularly suited for use to encapsulate allosteric effectors of hemoglobin, thereby reducing the affinity of erythrocytes for oxygen and improving the release of oxygen from erythrocytes in tissues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/US94/03189 filed Mar. 23, 1994 and is acontinuation-in-part of U.S. patent application Ser. No. 08/035,467,filed Mar. 23, 1993, now abandoned, which is hereby incorporated byreference.

TECHNICAL FIELD

The present invention relates to methods and apparatuses for theencapsulation of biologically-active substances in various cellpopulations. More particularly, the present invention relates to amethod and apparatus for the encapsulation of allosteric effectors ofhemoglobin in erythrocytes by electroporation to achieve therapeuticallydesirable changes in the physical characteristics of the intracellularhemoglobin.

BACKGROUND OF THE INVENTION

In the vascular system of an adult human being, blood has a volume ofabout 5 to 6 liters. Approximately one half of this volume is occupiedby cells, including red blood cells (erythrocytes), white blood cells(leukocytes), and blood platelets. Red blood cells comprise the majorityof the cellular components of blood. Plasma, the liquid portion ofblood, is approximately 90 percent water and 10 percent various solutes.These solutes include plasma proteins, organic metabolites and wasteproducts, and inorganic compounds.

The major function of red blood cells is to transport oxygen from thelungs to the tissues of the body, and transport carbon dioxide from thetissues to the lungs for removal. Very little oxygen is transported bythe blood plasma because oxygen is only sparingly soluble in aqueoussolutions. Most of the oxygen carried by the blood is transported by thehemoglobin of the erythrocytes. Erythrocytes in mammals do not containnuclei, mitochondria or any other intracellular organelles, and they donot use oxygen in their own metabolism. Red blood cells contain about 35percent by weight hemoglobin, which is responsible for binding andtransporting oxygen.

Hemoglobin is a protein having a molecular weight of approximately64,500. It contains four polypeptide chains and four heme prostheticgroups in which iron atoms are bound in the ferrous state. Normalglobin, the protein portion of the hemoglobin molecule, consists of twoα chains and two β chains. Each of the four chains has a characteristictertiary structure in which the chain is folded. The four polypeptidechains fit together in an approximately tetrahedral arrangement, toconstitute the characteristic quaternary structure of hemoglobin. Thereis one heme group bound to each polypeptide chain which can reversiblybind one molecule of molecular oxygen. When hemoglobin combines withoxygen, oxyhemoglobin is formed. When oxygen is released, theoxyhemoglobin is reduced to deoxyhemoglobin.

Delivery of oxygen to tissues depends upon a number of factorsincluding, but not limited to, the volume of blood flow, the number ofred blood cells, the concentration of hemoglobin in the red blood cells,the oxygen affinity of the hemoglobin and, in certain species, on themolar ratio of intraerythrocytic hemoglobins with high and low oxygenaffinity. The oxygen affinity of hemoglobin depends on four factors aswell, namely: (1) the partial pressure of oxygen; (2) the pH; (3) theconcentration of 2,3-diphosphoglycerate (DPG) in the hemoglobin; and (4)the concentration of carbon dioxide. In the lungs, at an oxygen partialpressure of 100 mm Hg, approximately 98% of circulating hemoglobin issaturated with oxygen. This represents the total oxygen transportcapacity of the blood. When fully oxygenated, 100 ml of whole mammalianblood can carry about 21 ml of gaseous oxygen.

The effect of the partial pressure of oxygen and the pH on the abilityof hemoglobin to bind oxygen is best illustrated by examination of theoxygen saturation curve of hemoglobin. An oxygen saturation curve plotsthe percentage of total oxygen-binding sites of a hemoglobin moleculethat are occupied by oxygen molecules when solutions of the hemoglobinmolecule are in equilibrium with different partial pressures of oxygenin the gas phase.

The oxygen saturation curve for hemoglobin is sigmoid. Thus, binding thefirst molecule of oxygen increases the affinity of the remaininghemoglobin for binding additional oxygen molecules. As the partialpressure of oxygen is increased, a plateau is approached at which eachof the hemoglobin molecules is saturated and contains the upper limit offour molecules of oxygen.

The reversible binding of oxygen by hemoglobin is accompanied by therelease of protons, according to the equation:

    HHb.sup.+ +O.sub.2 ⃡HbO.sub.2 +H.sup.+

Thus, an increase in the pH will pull the equilibrium to the right andcause hemoglobin to bind more oxygen at a given partial pressure. Adecrease in the pH will decrease the amount of oxygen bound.

In the lungs, the partial pressure of oxygen in the air spaces isapproximately 90 to 100 mm Hg and the pH is also high relative to normalblood pH (up to 7.6). Therefore, hemoglobin will tend to become almostmaximally saturated with oxygen in the lungs. At that pressure and pH,hemoglobin is approximately 98 percent saturated with oxygen. On theother hand, in the capillaries in the interior of the peripheraltissues, the partial pressure of oxygen is only about 25 to 40 mm Hg andthe pH is also relatively low (about 7.2 to 7.3). Because muscle cellsuse oxygen at a high rate thereby lowering the local concentration ofoxygen, the release of some of the bound oxygen to the tissue isfavored. As the blood passes through the capillaries in the muscles,oxygen will be released from the nearly saturated hemoglobin in the redblood cells into the blood plasma and thence into the muscle cells.Hemoglobin will release about a third of its bound oxygen as it passesthrough the muscle capillaries, so that when it leaves the muscle, itwill be only about 64 percent saturated. In general, the hemoglobin inthe venous blood leaving the tissue cycles between about 65 and 97percent saturation with oxygen in its repeated circuits between thelungs and the peripheral tissues. Thus, oxygen partial pressure and pHfunction together to effect the release of oxygen by hemoglobin

A third important factor in regulating the degree of oxygenation ofhemoglobin is the allosteric effector 2,3-diphosphoglycerate (DPG). DPGis the normal physiological effector of hemoglobin in mammalianerythrocytes. DPG regulates the oxygen-binding affinity of hemoglobin inthe red blood cells in relationship to the oxygen partial pressure inthe lungs. The higher the concentration of DPG in the cell, the lowerthe affinity of hemoglobin for oxygen.

When the delivery of oxygen to the tissues is chronically reduced, theconcentration of DPG in the erythrocytes is higher than in normalindividuals. For example, at high altitudes the partial pressure ofoxygen is significantly less. Correspondingly, the partial pressure ofoxygen in the tissues is less. Within a few hours after a normal humansubject moves to a higher altitude, the DPG level in the red blood cellsincreases, causing more DPG to be bound and the oxygen affinity of thehemoglobin to decrease. Increases in the DPG level of red cells alsooccur in patients suffering from hypoxia. This adjustment allows thehemoglobin to release its bound oxygen more readily to the tissues tocompensate for the decreased oxygenation of hemoglobin in the lungs. Thereverse change occurs when people acclimated to high altitudes anddescend to lower altitudes.

As normally isolated from blood, hemoglobin contains a considerableamount of DPG. When hemoglobin is "stripped" of its DPG, it shows a muchhigher affinity for oxygen. When DPG is increased, the oxygen bindingaffinity of hemoglobin decreases. A physiologic allosteric effector suchas DPG is therefore essential for the normal release of oxygen fromhemoglobin in the tissues.

While DPG is the normal physiologic effector of hemoglobin in mammalianred blood cells, phosphorylated inositols are found to play the samerole in the erythrocytes of some birds and reptiles. Although IHP isunable to pass through the mammalian erythrocyte membrane, it is capableof combining with hemoglobin of mammalian red blood cells at the bindingsite of DPG to modify the allosteric conformation of hemoglobin, theeffect of which is to reduce the affinity of hemoglobin for oxygen. Forexample, DPG can be replaced by inositol hexaphosphate (IHP), which iseven more potent than DPG in reducing the oxygen affinity of hemoglobin.IHP has a 1000-fold higher affinity to hemoglobin than DPG (R. E.Benesch et al., Biochemistry, Vol. 16, pages 2594-2597(1977)) andincreases the P50 of hemoglobin up to values of 96.4 mm, Hg at pH 7.4,and 37 degrees C. (J. Biol. Chem., Vol. 250, pages 7093-7098(1975)).

The oxygen release capacity of mammalian red blood cells can be enhancedby introducing certain allosteric effectors of hemoglobin intoerythrocytes, thereby decreasing the affinity of hemoglobin for oxygenand improving the oxygen economy of the blood. This phenomenon suggestsvarious medical applications for treating individuals who areexperiencing lowered oxygenation of their tissues due to the inadequatefunction of their lungs or circulatory system.

Because of the potential medical benefits to be achieved from the use ofthese modified erythrocytes, various techniques have been developed inthe prior art to enable the encapsulation of allosteric effectors ofhemoglobin in erythrocytes. Accordingly, numerous devices have beendesigned to assist or simplify the encapsulation procedure. Theencapsulation methods known in the art include osmotic pulse (swelling)and reconstitution of cells, controlled lysis and resealing,incorporation of liposomes, and electroporation. Current methods ofelectroporation make the procedure commercially impractical on a scalesuitable for commercial use.

The following references describe the incorporation of polyphosphatesinto red blood cells by the interaction of liposomes loaded with IHP:Gersonde, et al., "Modification of the Oxygen Affinity of IntracellularHaemoglobin by Incorporation of Polyphosphates into Intact Red BloodCells and Enhanced O2 Release in the Capillary System", Biblthca.Haemat., No. 46, pp. 81-92(1980); Gersonde, et al., "Enhancement of theO2 Release Capacity and of the Bohr-Effect of Human Red Blood Cellsafter Incorporation of Inositol Hexaphosphate by Fusion withEffector-Containing Lipid Vesicles", Origins of Cooperative Binding ofHemoglobin, (1982); and Weiner, "Right Shifting of Hb-O₂ Dissociation inViable Red Cells by Liposomal Technique," Biology of the Cell, Vol. 47,(1983).

Additionally, U.S. Pat. Nos. 4,192,869, 4,321,259, and 4,473,563 toNicolau et al. describe a method whereby fluid-charged lipid vesiclesare fused with erythrocyte membranes, depositing their contents into thered blood cells. In this manner it is possible to transport allostericeffectors such as inositol hexaphosphate into erythrocytes, where, dueto its much higher binding constant IHP replaces DPG at its binding sitein hemoglobin.

In accordance with the liposome technique, IHP is dissolved in aphosphate buffer until the solution is saturated and a mixture of lipidvesicles is suspended in the solution. The suspension is then subjectedto ultrasonic treatment or an injection process, and then centrifuged.The upper suspension contains small lipid vesicles containing IHP, whichare then collected. Erythrocytes are added to the collected suspensionand incubated, during which time the lipid vesicles containing IHP fusewith the cell membranes of the erythrocytes, thereby depositing theircontents into the interior of the erythrocyte. The modified erythrocytesare then washed and added to plasma to complete the product.

The drawbacks associated with the liposomal technique include poorreproducibility of the IHP concentrations incorporated in the red bloodcells and significant hemolysis of the red blood cells followingtreatment. Additionally, commercialization is not practical because theprocedure is tedious and complicated.

In an attempt to solve the drawbacks associated with the liposomaltechnique, a method of lysing and the resealing red blood cells wasdeveloped. This method is described in the following publication:Nicolau, et al., "Incorporation of Allosteric Effectors of Hemoglobin inRed Blood Cells. Physiologic Effects," Biblthca. Haemat., No. 51, pp.92-107, (1985). Related U.S. Pat. Nos. 4,752,586 and 4,652,449 to Roparset al. also describe a procedure of encapsulating substances havingbiological activity in human or animal erythrocytes by controlled lysisand resealing of the erythrocytes, which avoids the RBC-liposomeinteractions.

The technique is best characterized as a continuous flow dialysis systemwhich functions in a manner similar to the osmotic pulse technique.Specifically, the primary compartment of at least one dialysis elementis continuously supplied with an aqueous suspension of erythrocyteswhile the secondary compartment of the dialysis element contains anaqueous solution which is hypotonic with respect to the erythrocytesuspension. The hypotonic solution causes the erythrocytes to lyse. Theerythrocyte lysate is then contacted with the biologically activesubstance to be incorporated into the erythrocyte. To reseal themembranes of the erythrocytes, the osmotic and/or oncotic pressure ofthe erythrocyte lysate is increased and the suspension of resealederythrocytes is recovered.

In related U.S. Pat. Nos. 4,874,690 and 5,043,261 to Goodrich et al. arelated technique involving lyophilization and reconstitution of redblood cells is disclosed. As part of the process of reconstituting thered blood cells, the addition of various polyanions, including inositolhexaphosphate, is described. Treatment of the red blood cells accordingto the process disclosed results in a cell with unaffected activity.Presumably, the IHP is incorporated into the cell during thereconstitution process, thereby maintaining the activity of thehemoglobin.

In U.S. Pat. Nos. 4,478,824 and 4,931,276 to Franco et al. a secondrelated method and apparatus is described for introducing effectivelynon-ionic agents, including inositol hexaphosphate, into mammalian redblood cells by effectively lysing and resealing the cells. The procedureis described as the "osmotic pulse technique." In practicing the osmoticpulse technique, a supply of packed red blood cells is suspended andincubated in a solution containing a compound which readily diffusesinto and out of the cells, the concentration of the compound beingsufficient to cause diffusion thereof into the cells so that thecontents of the cells become hypertonic. Next, a trans-membrane ionicgradient is created by diluting the solution containing the hypertoniccells with an essentially isotonic aqueous medium in the presence of atleast on desired agent to be introduced, thereby causing diffusion ofwater into the cells with a consequent swelling and an increases inpermeability of the outer membranes of the cells. This "osmotic pulse"causes the diffusion of water into the cells and a resultant swelling ofthe cells which increase the permeability of the outer cell membrane tothe desired agent. The increase in permeability of the membrane ismaintained for a period of time sufficient only to permit transport ofleast one agent into the cells and diffusion of the compound out of thecells.

Polyanions which may be used in practicing the osmotic pulse techniqueinclude pyrophosphate, tripolyphosphate, phosphorylated inositols,2,3-diphosphoglycerate (DPG), adenosine triphosphate, heparin, andpolycarboxylic acids which are water-soluble, and non-disruptive to thelipid outer bilayer membranes of red blood cells.

The osmotic pulse technique has several shortcomings including low yieldof encapsulation, incomplete resealing, lose of cell content and acorresponding decrease in the life span of the cells. The technique istedious, complicated and unsuited to automation. For these reasons, theosmotic pulse technique has had little commercial success.

Another method for encapsulating various biologically-active substancesin erythrocytes is electroporation. Electroporation has been used forencapsulation of foreign molecules in different cell types including IHPred blood cells as described in Mouneimne, et al., "Stable rightwardshifts of the oxyhemoglobin dissociation curve induced by encapsulationof inositol hexaphosphate in red blood cells using electroporation,"FEBS, Vol. 275, No. 1, 2, pp. 117-120 (1990).

The process of electroporation involves the formation of pores in thecell membranes, or in any vesicles, by the application of electric fieldpulses across a liquid cell suspension containing the cells or vesicles.During the poration process, cells are suspended in a liquid media andthen subjected to an electric field pulse. The medium may beelectrolyte, non-electrolyte, or a mixture of electrolytes andnon-electrolytes. The strength of the electric field applied to thesuspension and the length of the pulse (the time that the electric fieldis applied to a cell suspension) varies according to the cell type. Tocreate a pore in a cell's outer membrane, the electric field must beapplied for such a length of time and at such a voltage as to create aset potential across the cell membrane for a period of time long enoughto create a pore.

Four phenomenon appear to play a role in the process of electroporation.The first is the phenomenon of dielectric breakdown. Dielectricbreakdown refers to the ability of a high electric field to create asmall pore or hole in a cell membrane. Once a pore is created, a cellcan be loaded with a biologically-active substances. The secondphenomenon is the dielectric bunching effect, which refers to the mutualself attraction produced by the placement of vesicles in a uniformelectric field. The third phenomenon is that of vesicle fusion. Vesiclefusion refers to the tendency of membranes of biological vesicles, whichhave had pores formed by dielectric breakdowns, to couple together attheir mutual dialectic breakdown sites when they are in close proximity.The fourth phenomenon is the tendency of cells to line up along one oftheir axis in the presence of high frequency electric fields. Thus,electroporation relates to the use in vesicle rotational prealignment,vesicle bunching and dielectric constant or vesicles for the purpose ofloading and unloading the cell vesicle.

Electroporation has been used effectively to incorporate allostericeffectors of hemoglobin in erythrocytes. In an article by Mouneimne, Yet al., "Stable Rightward Shifts of Oxyhemoglobin DisassociationConstant Induced by Encapsulation of Inositol Hexaphosphate in Red BloodCells Using Electroporation", FEBS, Vol. 275, No. 1, 2, pages 11-120.Mouneimne and his colleagues reported that fight shifts of thehemoglobin-oxygen dissociation in treated erythrocytes havingincorporated IHP can be achieved. Measurements at 24 and 48 hours afterloading with IHP showed a stable P₅₀ value indicating that resealing ofthe erythrocytes was permanent. Furthermore, it was shown that red bloodcells loaded with inositol hexaphosphate have a normal half life ofeleven days. However, the results obtained by Mouneimne and hiscolleagues indicate that approximately 20% of the retransfused cellswere lost within the first 24 hours of transfusion.

The electroporation methods disclosed in the prior art are not suitablefor processing large volumes of sample, nor use of a high or repetitiveelectric charge. Furthermore, the methods are not suitable for use in acontinuous or "flow" electroporation chamber. Available electroporationchambers are designed for static use only. Namely, processing of samplesby batch. Continuos use of a "static" chamber results in over heating ofthe chamber and increased cell lysis. Furthermore, the existingtechnology is unable to incorporate a sufficient quantity of IHP in asufficient percentage of the cells being processed to dramaticallychange the oxygen carrying capacity of the blood. In addition, the priorart methods require elaborate equipment and are not suited for loadingred blood cells of a patient on site. Thus, the procedure is timeconsuming and not suitable for use on a commercial scale. What is neededis a simple, efficient and rapid method for encapsulatingbiologically-active substances in erythrocytes while preserving theintegrity and biologic function of the cells. The potential therapeuticapplications of biologically altered blood cells suggests the need forsimpler, and more effective and complete methods of encapsulation ofbiologically-active substances, including allosteric effectors ofhemoglobin in intact erythrocytes.

There are numerous clinical conditions that would benefit fromtreatments that would increase tissue delivery of oxygen bound tohemoglobin. For example, the leading cause of death in the United Statestoday is cardiovascular disease. The acute symptoms and pathology ofmany cardiovascular diseases, including congestive heart failure,myocardial infarction, stroke, intermittent claudication, and sicklecell anemia, result from an insufficient supply of oxygen in fluids thatbathe the tissues. Likewise, the acute loss of blood followinghemorrhage, traumatic injury, or surgery results in decreased oxygensupply to vital organs. Without oxygen, tissues at sites distal to theheart, and even the heart itself, cannot produce enough energy tosustain their normal functions. The result of oxygen deprivation istissue death and organ failure.

Although the attention of the American public has long been focused onthe preventive measures required to alleviate heart disease, such asexercise, appropriate dietary habits, and moderation in alcoholconsumption, deaths continue to occur at an alarming rate. Since deathresults from oxygen deprivation, which in turn results in tissuedestruction and/or organ dysfunction, one approach to alleviate thelife-threatening consequences of cardiovascular disease is to increaseoxygenation of tissues during acute stress. The same approach is alsoappropriate for persons suffering from blood loss or chronic hypoxicdisorders, such as congestive heart failure.

Another condition which could benefit from an increase in the deliveryof oxygen to the tissues is anemia. A significant portion of hospitalpatients experience anemia or a low "crit" caused by an insufficientquantity of red blood cells or hemoglobin in their blood. This leads toinadequate oxygenation of their tissues and subsequent complications.Typically, a physician can temporarily correct this condition bytransfusing the patient with units of packed red blood cells.

Enhanced blood oxygenation may also reduce the number of heterologoustransfusions and allow use of autologous transfusions in more case. Thecurrent method for treatment of anemia or replacement of blood loss istransfusion of whole human blood. It is estimated that three to fourmillion patients receive transfusions in the U.S. each year for surgicalor medical needs. In situations where there is more time it isadvantageous to completely avoid the use of donor or heterologous bloodand instead use autologous blood.

Often the amount of blood which can be drawn and stored prior to surgerylimits the use of autologous blood. Typically, a surgical patient doesnot have enough time to donate a sufficient quantity of blood prior tosurgery. A surgeon would like to have several units of blood available.As each unit requires a period of several weeks between donations andcan not be done less than two weeks prior to surgery, it is oftenimpossible to sequester an adequate supply of blood. By processingautologous blood with IHP, less blood is required and it becomespossible to completely avoid the transfusion of heterologous blood.

As IHP-treated red cells transport 2-3 times as much oxygen as untreatedred cells, in many cases, a physician will need to transfuse fewer unitsof IHP-treaded red cells. This exposes the patient to less heterologousblood, decreases the extent of exposure to vital diseases from blooddonors and minimizes immune function disturbances secondary totransfusions. The ability to infuse more efficient red blood cells isalso advantageous when the patients blood volume is excessive. In othermore severe cases, where oxygen transport is failing, the ability torapidly improve a patient's tissue oxygenation is life saving.

Although it is evident that methods of enhancing oxygen delivery totissues have potential medical applications, currently there are nomethods clinically available for increasing tissue delivery of oxygenbound to hemoglobin. Transient, 6 to 12 hour elevations of oxygendeposition have been described in experimental animals using either DPGor molecules that are precursors of DPG. The natural regulation of DPGsynthesis in vivo and its relatively short biological half-life,however, limit the DPG concentration and the duration of increasedtissue PO₂, and thus limit its therapeutic usefulness.

Additionally, as reported in Genetic Engineering News, Vol. 12, No. 6,Apr. 15, 1992, several groups are attempting to engineer freeoxygen-carrying hemoglobin as a replacement for human blood.Recombinant, genetically modified human hemoglobin that does not breakdown in the body and that can readily release up to 30% of its boundoxygen is currently being tested by Somatogen, Inc., of Boulder Colo.While this product could be useful as a replacement for blood lost intraumatic injury or surgery, it would not be effective to increase PO₂levels in ischemic tissue, since its oxygen release capacity isequivalent to that of natural hemoglobin (27-30% ). As are allrecombinant products, this synthetic hemoglobin is also likely to be acostly therapeutic.

Synthetic human hemoglobin has also been produced in neonatal pigs byinjection of human genes that control hemoglobin production. This may bea less expensive product than the Somatogen synthetic hemoglobin, butproblems with oxygen affinity and breakdown of hemoglobin in the bodyare not solved by the method.

What is needed is a simple, efficient and rapid method for encapsulatingbiologically-active substances, such as IHP, in erythrocytes withoutdamaging the erythrocytes.

SUMMARY OF THE INVENTION

The present invention relates to a method and apparatus for theencapsulation of biologically-active substances in various cellpopulations. More specifically, the present invention provides anautomated, self-contained, flow apparatus for encapsulating allostericeffectors, such as inositol hexaphosphate, in red blood cells, therebyreducing the affinity of the hemoglobin for oxygen and enhancing thedelivery of oxygen by red blood cells to tissues. Encapsulation ispreferably achieved by electroporation; however, it is contemplated thatother methods of encapsulation may be used in practicing the presentinvention. The method and apparatus of the present invention is equallysuited to the encapsulation of a variety of biologically-activesubstances in various cell populations.

The apparatus and method of the present invention is suited to theincorporation of a variety of biologically-active substances in cellsand lipid vesicles. The method and apparatus of the present inventionmay be used for introducing a compound or biologically-active substanceinto a vesicle whether that vesicle is engineered or naturallyoccurring. For example, the apparatus and method of the presentinvention may be used to introduce IHP into erythrocytes.

The encapsulation of inositol hexaphosphate in red blood cells byelectroporation according to the present invention results in asignificant decrease in the hemoglobin affinity for oxygen withoutaffecting the life span, ATP levels, K+ levels, or normal rheologicalcompetence of the cells. In addition, the Bohr effect is not alteredexcept to shift the O₂ binding curve to the right. Lowering the oxygenaffinity of the erythrocytes increases the capacity of erythrocytes todissociate the bound oxygen and thereby improves the oxygen supply tothe tissues. Enhancement of the oxygen-release capacity of erythrocytesbrings about significant physiological effects such as a reduction incardiac output, an increase in the arteriovenous differences, andimproved tissue oxygenation.

The modified erythrocytes prepared in accordance with the presentinvention, having improved oxygen release capacities, may find their usein situations such as those illustrated below:

1. Under conditions of low oxygen-partial pressure, such as at highaltitudes;

2. When the oxygen exchange surface of the lung is reduced, such asoccurs in emphysema;

3. When there is an increased resistance to oxygen diffusion in thelung, such as occurs in pneumonia or asthma;

4. When there is a decrease in the oxygen-transport capacity oferythrocytes, such as occurs with erythropenia or anemia, or when anarteriovenous shunt is used;

5. To treat blood circulation disturbances, such as arteriosclerosis,thromboembolic processes, organ infarct or ischemia;

6. To treat conditions of high oxygen affinity of hemoglobin, such ashemoglobin mutations, chemical modifications of N-terminal amino acidsin the hemoglobin-chains, or enzyme defects in erythrocytes;

7. To accelerate detoxification processes by improving oxygen supply;

8. To decrease the oxygen affinity of conserved blood; or

9. To improve the efficacy of various cancer treatments.

According to the method and apparatus of the present invention, it ispossible to produce modified erythrocytes which contribute to animproved oxygen economy of the blood. These modified erythrocytes areobtained by incorporation of allosteric effectors, such as IHP, byelectroporation of the erythrocyte membranes.

The incorporation of the biologically-active substances into the cellsin accordance with the method of the present invention, including theencapsulation of allosteric effectors of hemoglobin into erythrocytes,is conducted extracorporally via an automated, flow electroporationapparatus. Briefly, a cell suspension is introduced into the separationand wash bowl chamber of the flow encapsulation apparatus. The cells areseparated from the suspension, washed and resuspended in a solution ofthe biologically-active substance to be introduced into the cell. Thissuspension is introduced into the electroporation chamber and thenincubated. Following electroporation and incubation, the cells arewashed and separated. A contamination check is optionally conducted toconfirm that all unencapsulated biologically-active substance has beenremoved. Then, the cells are prepared for storage or reintroduction intoa patient.

In accordance with the present invention and with reference to thepreferred embodiment, blood is drawn from a patient, the erythrocytesare separated from the drawn blood, the erythrocytes are modified by theincorporation of allosteric effectors and the modified erythrocytes andblood plasma is reconstituted. In this manner, it is possible to prepareand store blood containing IHP-modified erythrocytes.

The apparatus of the present invention provides an improved method forthe encapsulation of biologically-active substances in cells includingan apparatus which is self-contained and therefore sterile, an apparatuswhich can process large volumes of cells within a shortened time period,an apparatus having improved contamination detection, cooling andincubation elements, an apparatus is entirely automated and which doesnot require the supervision of a technician once a sample is introducedinto the apparatus.

Thus, it is an object of the present invention to provide an automated,continuous flow encapsulation apparatus.

It is a further object of the present invention to provide an automated,continuous flow electroporation apparatus.

It is a further object of the present invention to provide a continuousflow encapsulation apparatus which produces a homogenous population ofloaded cells or vesicles.

It is another object of the present invention to provide a continuousflow electroporation device which produces a homogenous population ofloaded cells or vesicles.

It is another object of the present invention to provide a sterile andnonpyrogenic method of encapsulating biologically-active substances incells.

It is another object of the present invention to provided a method andapparatus which results in stable resealing of cells or vesiclesfollowing electroporation to minimize lysis of the modified cells orvesicles after electroporation.

It is another object of the present invention to provide a flowencapsulation apparatus which produces a modified cell population fromwhich all exogenous non-encapsulated biologically-active substances havebeen removed.

It is another object of the present invention to provide anelectroporation apparatus which produces a modified cell population fromwhich all exogenous, non-encapsulated biologically-active substanceshave been removed.

It is another object of the present invention to provide a method andapparatus that allows continuous encapsulation of biologically-activesubstances in a population of cells or vesicles.

It is a further object of the present invention to provide a method andapparatus that achieves the above-defined objects, features, andadvantages in a single cycle.

It is another object of the present invention to provide a continuousflow electroporation chamber.

It is another object of the present invention to provide an improved andmore efficient method of encapsulating biologically active substances incells than those methods currently available.

It is a further object of the present invention to provide a populationof artificial cells suitable for medical use.

It is a further object of the present invention to provide a compositionsuitable for use in the treatment of conditions and/or disease statesresulting from a lack of or decrease in oxygenation.

Other objects, features, and advantages of the present invention willbecome apparent upon reading the following detailed description of thepreferred embodiment of the invention when taken in conjunction with thedrawing and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a first embodiment of a continuous flowencapsulation apparatus.

FIG. 2 is a schematic diagram of a second embodiment of a continuousflow encapsulation apparatus.

FIG. 3 is a top view of a first embodiment of the flow electroporationchamber with electrodes.

FIG. 4 is a top view of a first embodiment of the flow electroporationchamber without electrodes.

FIG. 5 is a side view of a first embodiment of the flow electroporationchamber.

FIG. 6 is an end view of a first embodiment of the flow electroporationchamber.

FIG. 7 is a side view of an electrode for use with the first embodimentof the flow electroporation chamber.

FIG. 8 is a front view of the electrode of FIG. 7.

FIG. 9 is an exploded perspective view of a second embodiment of theflow electroporation chamber.

FIG. 10 is a perspective view of the flow electroporation chamber ofFIG. 9 with the chamber being assembled.

FIG. 11 is a graph comparing the effect of various fieldstrengths, understatic or flow conditions, on the % oxygenation of IHP-encapsulated redblood cells.

FIG. 12 is a table comparing the effects of various fieldstrengths,under static or flow conditions, on the P₅₀ value of IHP-encapsulatedred blood cells.

FIG. 13 is a table comparing the survival rates of red blood cellssubjected to electroporation under static and flow conditions at variousfieldstrengths.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an automated, self-contained, flowapparatus for encapsulating allosteric effectors, such as inositolhexaphosphate, in red blood cells. The apparatus of the presentinvention combines the features of a plasmaphoresis apparatus with thoseof a flow electroporation apparatus to form an automated, self-containedflow electroporation device. The present invention further comprises anew flow electroporation chamber that allows use of the chamber underflow rather than static conditions. It is contemplated that the methodand apparatus of the present invention may be used to encapsulate avariety of biologically-active substances in diverse cell populations.

Additionally, the present invention provides a population of modifiedcells having physical characteristics that make the cells particularlyuseful for treating conditions which demand or benefit from an increasein the delivery of oxygen to the tissues. In accordance with the methodof the present invention, a homogenous population of IHP loaded redblood cells can be obtained with reduced contamination and a reducedpropensity to lyse following encapsulation. The treated red blood cellsexhibit normal life spans in circulation. Using the present invention,red blood cells of a patient in need of the treatment can be quicklyloaded and returned to the patient's circulation.

The method of operation of the apparatus of the present invention isdescribed below with reference to the preferred use of the apparatus,i.e., the encapsulation of allosteric effectors of hemoglobin in redblood cells. Inositol hexaphosphate is the preferred allosteric effectorto be used with the present invention. Other sugar phosphates, such asinositol pentaphosphate, inositol tetraphosphate, inositol triphosphate,inositol diphosphate and diphosphatidyl inositol diphosphate, can alsobe used. Other substances. Additionally, the apparatus maybe adapted toutilize methods of encapsulation other than electroporation. suitableallosteric effectors include polyphoshates such as nucleotidetriphosphates, nucleotide diphosphates, nucleotide monophosphates, andalcohol phosphate esters. In case of certain mutations of hemoglobin,e.g. "Zurich" hemoglobin, organic anions such as polycarboxylic acidscan be used as allosteric effectors. Finally, it is possible to useinorganic anions such as hexacyano ferrate, phosphate or chloride asallosteric effectors.

Red blood cells that have been loaded with inositol hexaphosphateaccording to the present invention can be used to treat a wide varietyof diseases and disease states. The IHP-loaded red blood cells madeaccording to the present invention can be administered to a patientundergoing a heart attack thereby increasing the oxygen delivery to theischemic heart tissue and, at the same time, reducing the cardiacoutput. The IHP-loaded red blood cells made according to the presentinvention also can be used to treat any ischemic condition including,but not limited to, stroke, diabetes, sickle cell disease, burns,intermittent claudication, emphysema, hypothermia, peripheral vasculardisease, disseminated intravascular coagulation, adult respiratorydistress syndrome (ARDS) and cystic fibrosis. A detailed description ofthe medical applications of compositions prepared in accordance with themethod of the present invention is also provided below.

Continuous Flow Encapsulation Apparatus

The method of operation of the apparatus of the present invention isdescribed below with reference to the preferred use of the apparatus,i.e., the encapsulation of allosteric effectors of hemoglobins in redblood cells by electroporation. It is to be understood that theapparatus may be adapted to accommodate other cell populations orvesicles, and other biologically active substances. Additionally, theapparatus maybe adapted to utilize methods of encapsulation other thanelectroporation.

Briefly, in accordance with the present invention, a sample of blood isintroduced into the continuos flow encapsulation apparatus. If red bloodcells are being collected, the blood can either be drawn directly from apatient or can be previously drawn blood. The blood is initiallyseparated into red blood cells, plasma and white blood cells, and wasteproducts. The waste products include the diluent and various bloodsolutes remaining in the supernatant after centrifugation. They arestored in a waste reservoir within the apparatus. The blood plasma andwhite blood cells are also retained in a reservoir within the systemwhile the red blood cells are admixed with the substance to beencapsulated. The suspension of red blood cells is then subjected toelectroporation. Following electroporation, the red blood cells areincubated under conditions which allow the cells to reseal. They arethen processed and washed to eliminate exogenous, non-encapsulatedbiologically-active substances. When the cells have been processed, thered blood cells containing the encapsulated substances can be optionallyreconstituted with the blood plasma and white blood cells. Thereconstituted blood may then be returned directly to the patient or canbe stored for later use. Although described as discrete steps, theprocess is essentially continuous.

A first embodiment of the present invention is described with referenceto FIG. 1, which schematically illustrates the structure of thecontinuous flow encapsulation apparatus of the present invention.

In accordance with the present invention, a volume of whole blood isadmitted into the electroporation system 5 at input 11. The blood samplemay optionally be drawn directly from a patient into the electroporationsystem 5, or the blood may be drawn at an earlier time and stored priorto introduction into the system 5. Valve 12 is opened to admit thesample into the system 5. Simultaneously, valve 25 is opened and pump 22is engaged to admit an anti-coagulant from the anti-coagulant reservoir27. A suitable anticoagulant is heparin, although other anticoagulantscan be used. The preferred anticoagulant is ACD. Valves 15 and 36 arealso opened and pump 40 is engaged. The admixture of anticoagulant andwhole blood passes through a filter 18 and a pressure evaluation system19 that monitors the flow through the apparatus, and is collected in ablood separation and wash bowl 44 which is activated when pump 40 isengaged. A sensor indicates when the blood separation and wash bowl 44has been filled with red blood cells. When it has been filled, the bloodsupply is stopped. The steps involving separation of the bloodcomponents can be accomplished by a plasmaphoresis apparatus, such asthe plasmaphoresis apparatus manufactured by Haemonetics Corporation(Haemonetics Corporation, Braintree, Mass.).

As explained above, when pump 40 is engaged in a clockwise direction,the blood separation and wash bowl 44 is engaged and the anti-coagulantand whole blood suspension is centrifuged to separate the plasma, whiteblood cells, red blood cells, and waste. Valve 87 is opened to admit theplasma and white blood cells into the plasma reservoir 89.

Optionally and dependent on the cell population being processed by theapparatus, the cells retained in the blood separation and was bowl 44are then washed. Valves 33, 15, and 36 are opened to admit saline bufferfrom the diluent reservoir 30 into the blood separation and wash bowl 44which contains the red blood cells. Pump 40 is still engaged. The redblood cells are then washed and centrifuged. The preferred saline bufferis a 0.9% sodium chloride solution, although other physiologicallyisotonic buffers can be used to dilute and wash the red blood cells.Valve 54 is opened to admit the waste into the waste reservoir 57 duringthe washing process. Again, the waste is stored in the waste reservoir57 and the red blood cells are retained in the blood separation and washbowl 44. The wash process is repeated if necessary.

Following separation of the red blood cells, pump 40 is reversed, pump22 is turned off, valves 12, 15, 33, 36, 25, 87, and 54 are closed, andvalves 97 and 64 are opened. The IHP solution is pumped out of the IHPreservoir 50 while, simultaneously, red blood cells are pumped out ofthe blood separation and wash bowl 44 towards the cooling coil 68. Thered blood cells and IHP solution are admixed in the tubing of theapparatus at junction 67 and then pumped through the cooling coil 68. Ina preferred embodiment of the present invention, and as explained indetail below, the IHP solution and red blood cells may be admixed in theseparation and wash bowl 44 before being admitted into the cooling coil68.

The preferred concentration of IHP in the solution is betweenapproximately 10 mMol and 100 mMol with a more preferred concentrationof approximately 23 to 35 mMol, and a most preferred concentration of 35mMol. The preferred IHP solution comprises the following compounds, inthe following concentrations:

35 mMol IHP salt neutralized with 35 mMol IHP acid

to a pH of 7.3

33 mMol K₂ HPO₄

7.0 mMol NaH₂

30.6 mMol KCL

6.4 mMol NaCl

7.3 mMol Sucrose

5.0 mMol ATP

A second IHP solution for use with the present invention comprises thefollowing compounds, in the following concentrations:

23 mMol IHP salt neutralized with HCl to a pH of 7.3

40 mMol K₂ HPO₄

7 mMol NaH₂

The IHP may be obtained from Sigma Chemical Company of St. Louis, Mo.

The hematocrit of the suspension is preferably between approximately 30and 80 with the most preferred hematocrit of approximately 50. Pump 40is designed to pump both red blood cells and IHP solution and can beadjusted so that the final hematocrit entering the cooling coil 68 canbe predetermined.

After mixing, the red blood cell-IHP suspension is then pumped through acooling coil 68. Cooling can be achieved with a water bath or with athermo-electric based cooling system. For example, cooling coil 68 isimmersed in a cooling bath in the cooling reservoir 69. When the redblood cell-IHP suspension passes through the cooling coil 68, thesuspension is cooled to a temperature of between approximately 1° C. and12° C., preferably approximately 4° C. Cooling the red blood cellsensures the survival of the pore created in the cell membrane during theelectroporation process. The use of a cooling coil aids in the speed ofcooling by increasing the surface area of the sample in contact with thecooling element. Optionally, the cooling coil can be surrounded by athermo-electric heat pump.

Certain applications may require heating of the cell suspension prior toelectroporation. In such a case, a heating coil may replace the coolingcoil 68. The maximum temperature tolerated by red blood cells isapproximately 37° C.

A thermoelectric heat pump works by extracting thermal energy from aparticular region, thereby reducing its temperature, and then rejectingthe thermal energy into a "heat sink" region of higher temperature. Atthe cold junction, energy is absorbed by electrons as they pass from alow energy level in the p-type semiconductor element, to a higher energylevel in the n-type semiconductor element. The power supply provides theenergy to move the electrons through the system. At the hot junction,energy is expelled into a heat sink as electrons move from a high energylevel element (n-type) to a lower energy level element (p-type).

Thermoelectric elements are totally solid state and do not have movingmechanical pans or require a working fluid, as do vapor-cycle devices.However, thermoelectric heat pumps perform the same cooling functions asfreon-based vapor compression or absorption refrigerators.Thermoelectric heat pumps are highly reliable, small in size andcapacity, low cost, low weight, intrinsically safer than many othercooling devices, and are capable of precise temperature control.

The preferred thermoelectric heat pumps for use in the present inventionare manufactured by MELCOR Materials Electronic Products Corp. ofTrenton, N.J. The thermocouples are made of high performance crystallinesemiconductor material. The semiconductor material is bismuth telluride,a quaternary alloy of bismuth, tellurium, selenium, and antimony, dopedand processed to yield oriented polycrystalline semiconductors withproperties. The couples, connected in series electrically and inparallel thermally, are integrated into modules. The modules arepackaged between metallized ceramic plates to afford optimum electricalinsulation and thermal conduction with high mechanical strength incompression. Modules can be mounted in parallel to increase the heattransfer effect or can be stacked in mullet-stage cascades to achievehigh differential temperatures. Passing a current through the heat pumpgenerates a temperature differential across the thermocouples, withmaximum ratings of 70° C. and higher.

After cooling, the red blood cell-IHP suspension enters theelectroporation chamber 72 where an electric pulse is administered froma pulse generator 75 to the red blood cell-IHP suspension, causingopenings to form within the cell membranes of the red blood cells.Optionally, an automatic detection system will turn the pulse generator75 on when the chamber 72 is filled with red blood cell-IHP suspension.An electrical pulse is applied to the suspension every time the chamber72 is filled with unencapsulated cells. A conventional electroporationchamber may be used when the operation of the apparatus is static,namely, when single discrete batches of cells are processed. In apreferred embodiment of the present invention a flow electroporationchamber is used. In one embodiment, a flow electroporation chamber 72 isconstructed of clear polyvinyl chloride, and contains two opposingelectrodes spaced a distance of 7 mm apart. The distance between theelectrodes will vary depending on the flow volume and fieldstrength.Preferably, the flow electroporation chamber 72 is disposable. Theelectroporation chamber may also be constructed of polysolfone, which ispreferably for use with certain sterilization procedures, such asautoclaving. A detailed description of the structure and construction ofthe flow electroporation chamber is provided below.

The red blood cell-IHP suspension passes between the two electrodes ofthe electroporation chamber 72. When a suspension of non-treated cellsenter the chamber 72, an electrical field of 1 to 3 KV/cm is created andmaintained for a period of 1 to 4 milliseconds, preferably for a periodof 2 milliseconds with a 1.8 ml flow chamber. Preferably, the IHP-redblood cell suspension is subjected to three high voltage pulses pervolume at a fieldstrength of approximately 2600 to 3200 V/cm per pulse.The pulse of current across the cell membranes causes an electricalbreakdown of the cell membranes, which creates pores in the membranes.IHP then diffuses into the cell through these pores.

Following electroporation, the red blood cell-IHP suspension enters anincubation chamber 78 where the suspension is incubated at roomtemperature for an incubation time of between approximately 15 minutesand 120 minutes with the preferred incubation time of 30 to 60 minutes.Optionally, the red blood cell-IHP suspension is incubated forapproximately 5 minutes at a temperature of approximately 37° C., and atleast 15 minutes at room temperature. The incubation chamber 78 mayoptionally be surrounded by a heating means 80. For example, the heatingmeans 80 can be a water bath or can be a thermoelectric heat pump.

Optionally, the incubator 78 contains a resealing buffer which aids inresealing and reconstitution of the red blood cells. The preferredcomposition of the resealing buffer is provided below:

    ______________________________________                                        RESEALING BUFFER                                                              ______________________________________                                        I. Combine                                                                    Sodium chloride  150        mMol                                              Potassium chloride                                                                             8          mMol                                              Sodium phosphate 6          mMol                                              Magnesium sulfate                                                                              2          mMol                                              Glucose          10         mMol                                              Adenine          1          mMol                                              Inosine          1          mMol                                              Penicillin G     500        units/ml                                          Chloramphenicol  0.1        mg/ml                                             II. Add                                                                       BSA              3.5%                                                         Calcium chloride 2          mMol                                              ______________________________________                                    

In the preferred embodiment of the present invention, no resealingbuffer is used.

Following incubation, valve 51 is opened and pump 40 is engaged and thered blood cell-IHP suspension is returned to the blood separation andwash bowl 44 from the incubation chamber 78. The excess IHP solution isremoved from the red blood cell suspension by centrifugation. The wasteHIP solution is directed to waste reservoir 57. Valves 33, 15 and 36 arethen opened to admit a volume of diluent into the blood separation andwash bowl 44. The red blood cell-IHP suspension is then centrifuged andthe supernatant is discarded in the waste reservoir 57 through valve 54leaving the red blood cells in the blood separation and wash bowl 44. Asaline buffer is added to the modified red blood cells from the diluentreservoir 30. The cells are washed and the supernatant is discardedfollowing centrifugation. The wash process is repeated if needed.

Optionally, as the waste is removed from the separation and wash bowl 44it passes through a contamination detector 46 to detect any free IHP inthe waste solution thereby confirming that exogenous non-encapsulatedIHP has been removed from the modified red blood cells. Thecontamination detection system relies on optical changes in the washingbuffer. After the modified red blood cells have been washed andcentrifuged, the supernatant passes through the contamination detector64 before it is deposited in the waste reservoir 57. If exogenous,non-encapsulated IHP remains in the washing buffer, The discardedsolution will be turbid. The turbidity is due to the reaction of IHPwith calcium, which is a component of the wash buffer. The contaminationdetector 46 uses an optical detection system. Preferably, the lightsource is an LED and the detector is a photodiode. The voltagedifference of the photodiode will indicate the amount of IHP in the washsolution. The contamination detector 46 is optional.

Following washing, the IHP-red blood cell product is optionallyreconstituted with the plasma and white blood cells which had beenretained in reservoir 89. The treated red blood cells may be collectedin a reinjection bag, either in a preservation media or in theautologous plasma of the patient.

The IHP-loaded red blood cells obtained can be administered directlyback into the patient or the cells can be stored for later use. The IHPin the red blood cells is not released during the normal storage time.

A preferred embodiment of the present invention is described withreference to FIG. 2, which schematically illustrates the structure ofthe continuous flow encapsulation apparatus of the present invention.Again, the method of operation of the apparatus is described withreference to the preferred use of the apparatus, i.e., the encapsulationof allosteric effectors of hemoglobin in red blood cells byelectroporation. It is to be understood that the apparatus may beadapted to accommodate other cell populations or vesicles, and otherbiologically active substances. Additionally, the apparatus maybeadapted to include other methods of encapsulation.

In accordance with the present invention, a sample of whole blood isadmitted into the electroporation system 10 at input 11. Valve 12 isopened to admit the sample into the system 10. Simultaneously, valve 25is opened and pump 22 is engaged to admit an anti-coagulant from theanti-coagulant reservoir 27. Valves 15 and 36 are also opened and pump40 is engaged.

The admixture of anticoagulant and whole blood passes through a filter18 and a pressure evaluation system 19, and is collected in a bloodseparation and wash bowl 44 which is activated when pump 40 is engaged.A sensor indicates when the blood separation and wash bowl 44 has beenfilled with red blood cells.

When pump 40 is engaged in a clockwise direction, the blood separationand wash bowl 44 is engaged and the anticoagulant and whole bloodsuspension is centrifuged to separate the plasma, white blood cells, redblood cells, and waste. Valve 87 is opened to admit the plasma and whiteblood cells into the plasma reservoir 89.

Optionally, the cells retained in the separation and wash bowl 44 arethen washed and centrifuged. Valves 33, 35, 15, and 36 are opened toadmit saline buffer from the diluent reservoir 30 into the bloodseparation and wash bowl 44 which contains the red blood cells. Valve 12is closed and pump 40 remains engaged.

During washing, valve 54 is opened to admit the waste into the wastereservoir 57 during the washing process. Again, the waste is stored inthe waste reservoir 57 and the red blood cells are retained in the bloodseparation and wash bowl 44. The wash process is repeated if necessary.A contamination detection system may optionally be installed between theseparation and wash bowl 44 and the waste reservoir 57 to control thewash process.

Following separation of the red blood cells, pump 40 is reversed, pump22 is turned off, valves 12, 15, 33, 35, 36, 25, 87, and 54 are closed,and valve 97 is opened. If the cells were washed, pump 22 was previouslyturned off and valves 12 and 25 had been closed. The IHP solution ispumped out of the IHP reservoir 50 and into the separation and wash bowl44 containing the red blood cells. There, the red blood cells and IHPare admixed to form a suspension.

The preferred concentration of IHP in the solution is betweenapproximately 10 mMol and 100 mMol with a more preferred concentrationof approximately 23 to 35 mMol, and with a most preferred concentrationof 35 mMol. The preferred IHP solution comprises the followingcompounds, in the following concentrations:

35 mMol IHP salt neutralized with 35 mMol IHP acid to a pH of 7.3

33 mMol K₂ HPO₄

7 mMol NaH₂

30.6 mMol KCL

6.4 mMol NaCl

7.3 mMol Sucrose

5.0 mMol ATP

The IHP may be obtained from Sigma Chemical Company of St. Louis, Mo.

The hematocrit of the suspension is preferably between approximately 30and 60 with the most preferred hematocrit of approximately 50. Pump 40is designed to pump both red blood cells and IHP solution and can beadjusted so that the final hematocrit entering the cooling coil 68 canbe predetermined.

The steps of collecting the specimen, separating the cells from thespecimen, washing the cells, and combining the cells with IHP can bedone with an apheresis-blood washing machine, such as that manufacturedby Haemonetics corporation. The apheresis-blood washing machine iscoupled to the flow electrophoresis apparatus described herein to form acontinuous flow electroporation apparatus.

After combining the red blood cells with the IHP solution, pump 40 isagain reversed, valve 97 is closed and valve 64 is opened. The red bloodcell-IHP suspension is then pumped through a thermoelectric cooling coil68. A blood bag from a blood warming set, such as the blood bag providedin the Fenwal® Blood Warming Set manufactured by Baxter HealthcareCorporation can be used as the cooling coil 68. When the red bloodcell-IHP suspension passes through the cooling coil 68 in the coolingreservoir 69, the suspension is cooled to a temperature of betweenapproximately 1° C. and 12° C., preferably approximately 4° C.Optionally, a pump may be added to the apparatus between the coolingcoil 68 and cooling reservoir 69, and the electroporation chamber 72, toensure a constant flow rate and compensate for fluctuation in volumethat occurs when the cooling coil 68 is filled.

Optionally, the pre-cooling step may be eliminated and the red bloodcell-IHP suspension may be directed to the electroporation chamber 72immediately after admixing. In such an instance, the cooling coil 68 andcooling reservoir 69 would be eliminated from the continuous flowencapsulation apparatus 10. Cooling prior to electroporation may not berequired if the temperature of the electroporation chamber issufficiently cool to maintain the cells suspension at 4° C.

After cooling, the red blood cell-IHP suspension enters theelectroporation chamber 72. The chamber 72 is maintained at atemperature of approximately 4° C. As the red blood cell-IHP suspensionpasses through the flow electroporation chamber 72, an electric pulse isadministered from a pulse generator 75 to the suspension causingopenings to form within the cell membranes of the red blood cells.

The red blood cell-IHP suspension passes between two electrodes of theelectroporation chamber 72. FIGS. 3 to 10 describe the electroporationchamber. In a preferred embodiment of the present invention, when asuspension of non-treated cells enters the chamber 72, the IHP-red bloodcell suspension is subjected to approximately three high voltage pulsesper volume at a fieldstrength of approximately 2600 to 3200 V/cm perpulse. The charge created across the cell membranes causes an electricalbreakdown of the cell membrane, which creates pores in the membrane. IHPthen diffuses into the cell through these pores.

During electroporation, an electrical field of 1 to 3 KV/cm is createdand maintained for a period of 1 to 4 milliseconds. The preferred pulselength is 3 to 4 milliseconds, with a most preferred pulse length of 2milliseconds. Pulse length is defined as 1/e. At a flow rate ofapproximately 10.6 ml/minute, the preferred number of pulses is 3, atthe preferred pulse rate of 0.29 Hz. The fieldstrength is defined as thevoltage over the distance between the electrodes. The distance betweenelectrodes is measured in centimeters. The preferred electricalparameters are as follows:

Exponential Pulse:

pulse length =1.5 to 2.5 ms

field strength =2.7 to 3 KV/cm

Following electroporation, the red blood cell-IHP suspension enters anincubation chamber 78 where the suspension is incubated at roomtemperature for an incubation time of between approximately 10 minutesand 120 minutes with a preferred incubation time of 30 minutes.Optionally, the red blood cell-IHP suspension is incubated forapproximately 5 minutes at a temperature of approximately 37° C., and atleast 15 minutes at room temperature. The incubation chamber 78 may besurrounded by a heating means 80. Any heating means 80 can be used inpracticing the present invention. The preferred heating means 80 are awater bath or a thermoelectric heat pump.

Optionally, the incubator 78 contains a resealing buffer which aids inresealing and reconstitution of the red blood cells. In the preferredembodiment of the present invention, no resealing buffer is used.

Following incubation, the red blood cell-IHP suspension is returned tothe blood separation and wash bowl 44 when valve 51 is opened and pump40 is engaged. The excess IHP solution is removed from the red bloodcell suspension by centrifugation. The waste IHP solution is directed towaste reservoir 57. Valves 33, 37, 15 and 36 are then opened to admit avolume of post wash solution from reservoir 31 into the blood separationand wash bowl 44. In a preferred embodiment of the present invention,the post wash solution comprises a 0.9% NaCl₂ solution, including 2.0 mMCaCl₂ and 2.0 mM MgCl₂. Any physiological saline may be used.

After addition of the post wash solution, the red blood cell-IHPsuspension is then centrifuged and the supernatant is discarded in thewaste reservoir 57 through valve 54 leaving the red blood cells in theblood separation and wash bowl 44. The wash process is repeated untilall unencapsulated IHP has been removed.

Optionally, as the waste is removed from the separation and wash bowl 44it passes through a contamination detector 46 to detect any free IHP inthe waste solution thereby confirming that exogenous non-encapsulatedIHP has been removed from the modified red blood cells. Thecontamination detector 46 is optional.

Following washing, the red blood cells containing IHP may bereconstituted with the plasma and white blood cells retained inreservoir 89. Pump 40 is engaged and valves 87, 36, and 92 are opened.The modified red blood cells and plasma and whim blood cells are pumpedto reservoir 96. A filter may be installed between reservoir 96 andvalve 92 to remove any aggregates or other impurities from thereconstituted modified blood.

The IHP-loaded red blood cells obtained in accordance with the method ofthe present invention can be administered directly back into the patientor the cells can be stored for later use. The IHP in the red blood cellsis not released during the normal storage time.

It is contemplated that continuous flow encapsulation apparatus of thepresent invention may be modified to utilize other encapsulationmethods.

Furthermore, it is contemplated that the continuous flow encapsulationapparatus may be adapted to process various diverse cell populations.Furthermore, the apparatus may be used to encapsulate biologicallyactive substances in artificial vesicles.

It is also contemplated that the continuous flow encapsulation apparatusof the present invention may be use to encapsulate a broad range ofbiologically active substances.

The flow electroporation apparatus of the present invention may beseparated from the plasmaphoresis apparatus of the present invention.The blood cooling system, peristaltic pump, electroporation chamber,pulse generator, and electronics comprising the flow electroporationapparatus may be linked to a plasmaphoresis apparatus and interface withthe controls of that machine.

While this invention has been described in specific detail withreference to the disclosed embodiments, it will be understood that manyvariations and modifications may be effected within the spirit and scopeof the invention as described in the appended claims.

Flow Electroporation Chamber

During electroporation, the insertion rate of IHP is linearly dependenton the voltage administered to the cells. Generally, the higher thevoltage, the more IHP is encapsulated; however, cell lysis is alsoincreased and cell survival is decreased. The efficiency of anelectroporation system may be judged by cell survival afterelectroporation. Poor cell survival indicates very low efficiency. Theamplitude and duration of the electrical pulse is responsible for theelectric breakdown of the cell membrane and creates pores in the polecaps parallel to the electric field. Thus, the factors to be consideredin designing an electroporation system include the field strength, thepulse length and the number of pulses.

A perfect electroporation target is shaped like a sphere, so itsorientation does not effect the efficiency of the applied field. Whenthe target is spherical, a single pulse with an field strength above thethreshold can electroplate 100% of the target. Red blood cells are diskshaped. Because of their shape and orientation in the electroporationchamber, only approximately 40% of the cells are electroplated during asingle pulse. To also electroporate the other 60%, the fieldstrength canbe increased. This increases the stress on the red blood cells in properorientation to the electric field and leads to lower survival rates ofthe cells.

To achieve more efficient encapsulation while reducing the incidence ofcell lysis and death, a flow electroporation chamber utilizing shortduration multiple pulses was developed. With the flow-through ratesteady and a steady field voltage, it was determined that plurality ofpulses would insert maximal quantities of IHP with minimal 2 to 24 hourlysis. A multiple-pulse system allows an increase in the cell survivalrate without increasing the field strength. When a multiple-pulse systemis used, orientation of the cells is not as critical as it is when asystem is a single pulse system is used. The lower field strength ismuch more gentle to the red blood cells. It is much easier toelectroporate every single cell in the multiple pulse system, becausethe timing between the flow rate of the red blood cells through thechamber and the electroporation pulses, and the orientation of the cellsis not as crucial as in a single pulse system. The flow multiple-pulseelectroporation system also increases both the short term and the longterm survival of red blood cells when compared to the single pulsemethod.

FIGS. 11 to 13 illustrate the effects of various field strengths, understatic or flow conditions, on the % oxygenation of IHP-encapsulated redblood cells over a range oxygen pressures; on the P₅₀ value ofIHP-encapsulated red blood cells (two concentrations of IHP solutionswere compared); and, on the survival rates of red blood cells subjectedto electroporation. All readings were taken 24 hours afterelectroporation. The results indicated that multiple pulses atcomparatively low fieldstrengths produce optimal encapsulation results.

A cooled electroporation chamber is preferred to keep the red bloodcells at a constant temperature during the electroporation process,thereby enhancing their survival rates. This is accomplished by removingthe excess heat created by the electrical pulse during theelectroporation process. The excess heat may be removed either bycooling the electrodes or cooling the entire flow electroporationchamber. In accordance with the preferred embodiment of the presentinvention, the electrodes themselves are cooled.

During the electroporation process, blood is pumped through an inlet inthe electroporation chamber and the red blood cells are subject to aseries of electrical pulses as they travel through the chamber. Theyexit out the other end of the chamber. The chamber can be made of anytype of insulating material, including but not limited to ceramic,teflon, plexiglass, glass, plastic, silicon, rubber or other syntheticmaterials. Preferably, the chamber is comprised of glass or polysulfone.Whatever the composition of the chamber, the internal surface of thechamber should be smooth to reduce turbulence in the fluid passingthrough it. The housing of the chamber should be non-conductive andbiologically inert. In commercial use, it is anticipated that thechamber will be disposable.

In a preferred embodiment of the present invention, the electrodes thatcomprise part of the electroporation apparatus can be constructed fromany type of electrically or thermally conductive hollow stock material,including but not limited to brass, stainless steel, gold platedstainless steel, gold plated glass, gold plated plastic, or metalcontaining plastic. Preferably, the surface of the electrode is goldplated. Gold plating serves to eliminate oxidation and reduces thecollection of hemoglobin and other cell particles at the electrodes. Thesurface of the electrodes should be smooth.

The electrodes can be hollow, to allow cooling by liquid or gas, or theelectrodes can be solid, to allow for thermoelectric or any other typeof conductive cooling. Cooling could also be accomplished by cooling theelectroporation chamber itself, apart from cooling the electrodes.

Preferably, the flow electroporation chamber is disposable. A detaileddescription of two embodiments of the electroporation chamber of thepresent invention is provided below.

In one embodiment, the flow electroporation chamber is constructed ofclear polyvinyl chloride, and contains two opposing electrodes spaced adistance of approximately 7 mm apart. The electroporation chamber is amodification of a chamber obtained from BTX Electronic Company of SanDiego, Calif. However, when the electroporation chamber is usedcontinuously, it overheats and the survival rate of the cells processedby the apparatus decreases over time. To correct the overheating problemthat occurred when the apparatus was used in a continuous flow manner, acontinuous flow electroporation chamber was designed. A detaileddescription of the structure of the continuous flow electroporationchamber is provided below.

FIGS. 3 through 8 show one embodiment of the flow electroporationchamber 72 of the present invention. As can be seen in FIG. 3, the flowelectroporation chamber 72 includes a housing 100 having two electrodes102 inset on opposing sides of the housing 100 of the electroporationchamber 72. The housing 100 includes an inlet channel 104 at one end andan outlet channel 106 at the other. The inlet 104 and outlet 106channels include connectors 108 and 109 respectively, preferably of themale Luer variety. The connectors 108 and 109 are hollow and form theinlet 104 and outlet 106 channels into the interior of theelectroporation chamber 72.

As seen in FIGS. 4 and 5, an internal chamber 110 extends most of thelength of the housing 100 and is sized to receive the two electrodes102. The internal chamber 110 includes beveled surfaces 111 forreceiving the internal edges of the electrodes 102. The internal chamber110 is thus formed by the internal surfaces of the electrodes 102 andthe internal surfaces of the housing 100. The internal chamber 110 isconnected to the inlet 104 and outlet 106 channels.

As can be seen in FIGS. 7 and 8, the electrodes 102 of theelectroporation chamber 72 of FIGS. 3 to 6 are comprised of flat,elongated, hollow shells. The electrodes 102 include cooling inlets 112and cooling outlets 114 at their ends. As described above, the rearsurfaces of the electrodes 102, or the surface to the left in FIG. 7,fits flush against the beveled surface 111 of the housing 100.

The electroporation chamber 72 is designed such that the cell suspensionto be subjected to electroporation enters the electroporation chamber 72through the inlet 104 and expands to fill the internal chamber 110. Asthe red blood cell suspension flows through the internal chamber 110 apulse or charge is administered across the width of the internal chamber110.

To maintain a relatively constant temperature during the electroporationprocess, cooling fluid or cooling gas is pumped in the cooling inlet 112and out the cooling outlet 114 so that the electrodes 102 are maintainedat approximately 4° C.

FIGS. 9 and 10 display a second embodiment of the flow electroporationchamber 172. As can be seen in FIGS. 9 and 10, the flow electroporationchamber 172 includes a hollow housing 200 substantially rectangular inshape. Two electrodes 202 are inserted into the interior of the housing200 directly opposite one another, flush against the housing 200 walls.The flow electroporation chamber 172 further comprises an inlet channel204 at one end and an outlet channel 206 at the other end of the housing200. The inlet 204 and outlet 206 channels include connectors 208 and209 which are attached by tubing 216 to a cell suspension supply thatsupplies the cell suspension, i.e. the IHP-red blood cell suspension, tothe electroporation chamber 172. The connectors 208 and 209 and inlet204 and outlet 206 channels serve to direct the cell suspension into andout of the housing 200.

As can be seen in FIG. 10, one end of the inlet channel 204 and one endof the outlet channel 206 extends into the interior of the housing 200forming an internal chamber 210. The internal chamber 210 is thus formedby the internal surfaces of the electrodes 202, the internal surfaces ofthe housing 200 and the internal surfaces of the of the inlet 204 andoutlet 206 channels.

As can be seen in FIGS. 9 and 10, the electrodes 202 of the flowelectroporation chamber 172 comprise flat, elongated, hollow shells. Theelectrodes 202 include cooling inlets 212 and cooling outlets 214 attheir ends, through which a gas or fluid may be pumped through theelectrodes 202 to maintain a constant temperature duringelectroporation. The electrodes 202 are connected to a pulse generatorby cables 220.

As with the chamber described above, the electroporation chamber 172 ofFIGS. 9 and 10 is designed such that the suspension to be subjected toelectroporation enters the electroporation chamber 172 through the fluidinlet 204 and expands to fill the internal chamber 210. As the red bloodcells suspension flows through the internal chamber 210, a pulse orcharge is administered across the width of the internal chamber 210between the electrodes 202. To maintain a relatively constanttemperature during the electroporation process, cooling fluid or coolinggas is pumped in the cooling inlet 212 and out the cooling outlet 214 ofthe electrodes 202 through the connectors 208 and 209 so that theelectrodes 202 are maintained at approximately 4° C. It is also possiblethat the inlet channel 204, outlet channel 206 and connectors 208 and209 can be made as a solidly integrated glass part, rather than separatecomponents.

It is contemplated that the flow electroporation chamber 172 maybeconstructed from drawn glass or any other highly polished material. Itis preferable that the interior surface of the electroporation chamber172 be as smooth as possible to reduce the generation of surfaceturbulence. Drawn glass components are highly consistent with perfectsurface finishes. Furthermore, they are stable and inert to bloodcomponents. They are also relatively inexpensive, which is desirable fora disposable electroporation chamber.

The electrodes may also be comprised of drawn glass, electroplated withcolloidal gold. Again, the surfaces of the electrodes should be highlyfinished, highly conductive, yet biologically inert. Gold electroplateis durable and inexpensive. Fluidic connection can be accomplished usingcommonly available parts.

The flow electroporation chamber may be constructed either as a part ofthe entire flow encapsulation apparatus, or as an individual apparatus.The flow electroporation apparatus may then be connected to acommercially available plasmaphoresis machine for encapsulation ofparticular cell populations. For example, the flow electroporationchamber may be connected to commercially available plasmaphoresisequipment by electronic or translational hardware or software.Optionally, a pinch-valve array and controller driven by a PC programcan also be used to control the flow electroporation apparatus.Similarly, current power supplies are capable of establishing the powerlevels needed to run the flow electroporation chamber or flowencapsulation apparatus.

While this invention has been described in specific detail withreference to the disclosed embodiments, it will be understood that manyvariations and modifications may be effected within the spirit and scopeof the invention as described in the appended claims.

Application of IHP Treated Red Blood Cells

The present invention provides a novel method for increasing theoxygen-carrying capacity of erythrocytes. In accordance with the methodof the present invention, the IHP combines with hemoglobin in a stableway, and shifts its oxygen releasing capacity. Erythrocytes withIHP-hemoglobin can release more oxygen per molecule than hemoglobinalone, and thus more oxygen is available to diffuse into tissues foreach unit of blood that circulates. Under ordinary circumstances, IHP istoxic and cannot be tolerated as an ordinary drug. Attachment of IHP tohemoglobin in this novel procedure, however, neutralizes its toxicity.In the absence of severe chronic blood loss, treatment with acomposition prepared in accordance with the present method could resultin beneficial effects that persist for approximately ninety days.

Another advantage of IHP-treated red blood cells is that they do notlose the Bohr effect when stored. Normal red blood cells that have beenstored by conventional means do not regain their maximum oxygen carryingcapacity for approximately 24 hours. This is because the DGP in normalred blood cells diffuses away from the hemoglobin molecule duringstorage and must be replaced by the body after transfusion. In contrast,red blood cells treated according to the present invention are retaintheir maximum oxygen carrying capacity during storage and therefore candeliver maximum oxygen to the tissues immediately after transfusion intoa human or animal.

The uses of IHP-treated RBC's is quite extensive including the treatmentof numerous acute and chronic conditions including, but not limited to,hospitalized patients, cardiovascular operations, chronic anemia, anemiafollowing major surgery, coronary infarction and associated problems,chronic pulmonary disease, cardiovascular patients, autologoustransfusions, as an enhancement to packed red blood cells transfusion(hemorrhage, traumatic injury, or surgery). congestive heart failure,myocardial infarction (heart attack), stroke, peripheral vasculardisease, intermittent claudication, circulatory shock, hemorrhagicshock, anemia and chronic hypoxia, respiratory alkalemia, metabolicalkalosis, sickle cell anemia, reduced lung capacity caused bypneumonia, surgery, pneumonia, trauma, chest puncture, gangrene,anaerobic infections, blood vessel diseases such as diabetes, substituteor complement to treatment with hyperbaric pressure chambers,intra-operative red cell salvage, cardiac inadequacy, anoxia-secondaryto chronic indication, organ transplant, carbon monoxide, nitric oxide,and cyanide poisoning.

Treating a human or animal for any one or more of the above diseasestates is done by transfusing into the human or animal betweenapproximately 0.5 and 6 units (1 unit=500 ml) of IHP-treated blood thathas been prepared according to the present invention. In certain cases,there may be a substantially complete replacement of all the normalblood in a patient with IHP-treated blood. The volume of IHP-treated redblood cells that is administered to the human or animal will depend uponthe indication being treated. In addition, the volume of IHP-treated redblood cells will also depend upon concentration of IHP-treated red bloodcells in the red blood cell suspension. It is to be understood that thequantity of IHP red blood cells that is administered to the patient isnot critical and can vary widely and still be effective.

IHP-treated packed RBC's are similar to normal red blood cells in everycategory except that the IHP-treated packed red blood cells can deliver2 to 3 times as much oxygen to tissue per unit. A physician wouldtherefore chose to administer a single unit of IHP-treated packed redblood cells rather than 2 units of the normal red blood cells.IHP-treated packed red blood cells could be prepared in blood processingcenters analogously to the present blood processing methods, except forthe inclusion of a processing step where the IHP is encapsulated in thecells.

While this invention has been described in specific detail withreference to the disclosed embodiments, it will be understood that manyvariations and modifications may be effected within the spirit and scopeof the invention as described in the appended claims.

What is claimed is:
 1. A continuous flow apparatus for encapsulating abiologically-active substance into red blood cells comprising:a.introduction means for introducing blood into the apparatus; b.separating means in fluid communication with the introduction means forseparating the red blood cells from at least some matter within theblood; c. mixing means in fluid communication with the separating meansfor mixing the red blood cells with a solution of a biologically-activesubstance to provide a red blood cell suspension; d. electroporationmeans in fluid communication with the mixing means for subjecting thered blood cell suspension to electroporation, thereby causing thebiologically-active substance to be encapsulated in the red blood cells;e. incubating means in fluid communication with the electroporationmeans for incubating the red blood cells after electroporation toprovide modified red blood cells; and f. washing means in fluidcommunication with the incubating means for washing the modified redblood cells to remove unencapsulated biologically-active substancetherefrom.
 2. The apparatus of claim 1, further comprising a coolingmeans in fluid communication with the separating means and theelectroporation means for cooling the red blood cell suspension prior toelectroporation.
 3. The apparatus of claim 1, wherein the incubatingmeans further comprises a means for heating the red blood cellsintroduced into the incubating means.
 4. The apparatus of claim 1,further comprising a detection means in fluid communication with thewashing means for detecting extracellular biologically-active substance.5. The apparatus of claim 1, wherein the separating means is capable ofseparating plasma and leukocytes from the red blood cells.
 6. Theapparatus of claim 1, further comprising a collecting means in fluidcommunication with the washing means for storing the modified red bloodcells.
 7. A method of incorporating a biologically-active substance intored blood cells in a continuous flow system comprising the steps of:a.introducing blood into the continuous flow system; b. separating redblood cells from at least some plasma and leukocytes in the blood toprovide a plasma and leukocyte fraction; c. mixing the red blood cellswith a biologically-active substance to provide a red blood cellsuspension; d. electroporating the red blood cell suspension, therebycausing the biologically-active substance to be encapsulated in the redblood cells; e. incubating the red blood cells to allow the red bloodcells to reseal to provide modified red blood cells; and f. washing themodified red blood cells to remove unencapsulated biologically-activesubstance therefrom.
 8. The method of claim 7, further comprising beforethe step of electroporating the red blood cell suspension, the step ofcooling the red blood cell suspension.
 9. The method of claim 7, furthercomprising the step of heating the red blood cell suspension during theincubating step.
 10. The method of claim 7, further comprising followingthe step of washing the modified red blood cells, the step of detectingextracellular biologically-active substance.
 11. The method of claim 7,further comprising following the step of separating at least some plasmaand leukocytes from the blood, the step of storing the plasma andleukocyte fraction.
 12. The method of claim 7, further comprisingfollowing the step of washing the modified red blood cells, the step ofrestoring plasma and leukocytes to the modified red blood cells.
 13. Themethod of claim 7, wherein the biologically-active substance is inositolhexaphosphate.
 14. A red blood cell containing a biologically-activesubstance prepared in accordance with the method of claim
 13. 15. A redblood cell containing a biologically-active substance prepared inaccordance with the method of claim
 7. 16. A continuous flow apparatusfor encapsulation of a biologically-active substance into cells byelectroporation comprising:a. introduction means for introducing thecells into the apparatus; b. separating means in fluid communicationwith the introduction means for isolating the cells from at least somematter with which the cells are associated; c. mixing means in fluidcommunication with the separating means for mixing the cells with asolution of a biologically-active substance to provide a cellsuspension; d. electroporation means in fluid communication with themixing means for subjecting the cell suspension to electroporationthereby causing the biologically-active substance to be encapsulated inthe cells; e. incubating means in fluid communication with theelectroporation means for incubating the cells after electroporation toprovide modified cells; and f. washing means in fluid communication withthe incubating means for washing the modified cells to removeunencapsulated biologically-active substance therefrom.
 17. Theapparatus of claim 16, further comprising a cooling means in fluidcommunication with the separating means and the electroporation meansfor cooling the cell suspension.
 18. The apparatus of claim 16, whereinthe incubating means further comprises a means for heating the cellsintroduced into the incubating means.
 19. The apparatus of claim 16,further comprising a detection means in fluid communication with thewashing means for detecting unencapsulated biologically-activesubstance.
 20. The apparatus of claim 16, further comprising acollecting means in fluid communication with the washing means forstoring washed, modified cells.
 21. A method of incorporating abiologically-active substance into cells in a continuous flow systemcomprising the steps of:a. introducing the cells into the continuousflow system; b. isolating the cells from at least some matter with whichthe cells are associated; c. mixing the cells with a biologically-activesubstance to provide a cell suspension; d. electroporating the cellsuspension, thereby causing the biologically-active substance to beencapsulated in the cells; e. incubating the cells to allow the cells toreseal to provide modified cells; and f. washing the modified cells toremove unencapsulated biologically-active substance therefrom.
 22. Themethod of claim 21, further comprising the step of cooling the cellsuspension before electroporating the cell suspension.
 23. The method ofclaim 21, further comprising the step of heating the cell suspensionduring the incubating step.
 24. The method of claim 21, furthercomprising the step of detecting extracellular biologically-activesubstance after washing the modified cells.
 25. The method of claim 21,further comprising the step of collecting the modified cells afterwashing the modified cells.
 26. A cell containing a biologically-activesubstance prepared in accordance with the method of claim
 21. 27. Amethod of treating a disease comprising the step of administering to ahuman or animal a suspension of red blood cells treated according toclaim
 7. 28. A device comprising:a housing having internal wallsdefining a continuous internal chamber through said housing, saidinternal chamber being configured to receive a continuous flow volume ofblood therethrough; a first electrode having a first specified lengthand disposed within said internal chamber; a second electrode having asecond specified length and disposed within said internal chamber inspaced-apart relation to said first electrode; said first and secondelectrodes being arranged within said internal chamber to permit saidcontinuous flow volume of blood flowing through said internal chamber topass between said electrodes; and at least one of said first and secondelectrodes defining a cooling passage therethrough, said cooling passagebeing configured to receive a coolant therethrough for cooling said atleast one of said first and second electrodes.
 29. The device of claim28, wherein the first electrode and the second electrode each form atleast part of the internal walls which define the internal chamber. 30.The device of claim 28, wherein each of said first and second electrodesdefines a cooling passage therethrough, said cooling passages in saidfirst and second electrodes being configured to receive a coolanttherethrough for cooling said first and second electrodes.
 31. A methodof electroporating blood cells, comprising the steps of:a. providing adevice comprising:i. a housing having internal walls defining acontinuous internal chamber through said housing, said internal chamberbeing configured to receive a continuous flow volume of bloodtherethrough; ii. a first electrode having a first specified length anddisposed within said internal chamber; iii. a second electrode having asecond specified length and disposed within said internal chamber inspaced-apart relation to said first electrode; iv. said first and secondelectrodes being arranged within said internal chamber to permit saidcontinuous flow volume of blood flowing through said internal chamber topass between said electrodes; and v. at least one of said first andsecond electrodes defining a cooling passage therethrough; b. passing acontinuous flow volume of blood through said internal chamber andbetween said electrodes at a specified flow rate; c. causing pulses ofelectronic energy at a specified frequency to be emitted from said firstelectrode through the blood to said second electrode as said continuousflow volume of blood flows through said internal chamber, said first andsecond specified lengths of said electrodes, said specified flow rate ofsaid blood, and said specified frequency of said electronic energy beingsuch that blood cells in said volume of blood may be electroporatedwhile flowing through said internal chamber; and d. cooling said atleast one of said first and second electrodes by passing a coolantthrough said cooling passage thereof.
 32. The method of claim 31,wherein said step of providing a device comprising at least one of saidfirst and second electrodes defining a cooling passage therethroughcomprises the step of providing a device comprising both of said firstand second electrodes defining cooling passages therethrough, andwherein said step of cooling said at least one of said first and secondelectrodes comprises the step of cooling both of said first and secondelectrodes by passing a coolant through said cooling passages thereof.